Figures

Abstract

Background

Adult mesenchymal stem cells (MSCs) can be maintained over extended periods
of time before activation and differentiation. Little is known about the
programs that sustain the survival of these cells.

Principal Findings

Undifferentiated adult human MSCs (hMSCs) did not undergo apoptosis in
response to different cell death inducers. Conversely, the same inducers can
readily induce apoptosis when hMSCs are engaged in the early stages of
differentiation. The survival of undifferentiated cells is linked to the
expression of Bcl-Xl and Bcl-2 in completely opposite ways. Bcl-Xl is
expressed at similar levels in undifferentiated and differentiated hMSCs
while Bcl-2 is expressed only in differentiated cells. In undifferentiated
hMSCs, the down-regulation of Bcl-Xl is associated with an increased
sensitivity to apoptosis while the ectopic expression of Bcl-2 induced
apoptosis. This apoptosis is linked to the presence of cytoplasmic Nur 77 in
undifferentiated hMSCs.

Significance

In hMSCs, the expression of Bcl-2 depends on cellular differentiation and can
be either pro- or anti-apoptotic. Bcl-Xl, on the other hand, exhibits an
anti-apoptotic activity under all conditions.

Funding: This work was supported by grants from INSERM (Insitut National de la
Santé et la Recherche Médicale) and a special grant from the
“Ligue Nationale contre le Cancer” (programme Equipe
Labellisée). The funders had no role in study design, data collection and
analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Self-renewal and limited proliferation are the processes that allow the maintenance
of the stem cell pools throughout life [1]. Stem cells must thus maintain their molecular blueprints
over long periods of time and this quality control is achieved, in part, through the
induction of cell death in damaged cells [2]. Mesenchymal stem cells or bone
marrow stromal cells (MSCs) are committed to mesenchymal cell lineages such as bone,
cartilage, tendon, ligament, adipocytes and muscle; and possibly to other cell types
such as neurons [3]–[5]. MSCs are also essential for the proliferation and
differentiation of hematopoietic cells within the bone marrow compartment [3]–[5]. Beyond their
transdifferentiation process, MSCs are also involved in tissue repair and recently
have been considered as an ideal therapeutic vehicle in many diseases [6]–[7].

One important feature of MSCs is their capacity to survive over long periods of time
under homologous conditions but to die rapidly upon their transfer into another
individual [3]–[5]. These observations suggest that these cells, which are
highly proliferative in vitro, also possess an efficient cell death
machinery. There is little information about the mechanism of survival of most adult
stem/progenitor cells, although this question must be crucial with regards to their
physiological role(s). Apoptosis is a cell death program that is instrumental in
fetal and adult tissue homeostasis. It has been shown that fetal MSCs exhibit
functional apoptotic pathways [8]. In agreement with the latter hypothesis, genetically
modified rat MSCs containing an anti-apoptotic Bcl-2 gene exhibited enhanced cell
survival upon intracardiac engraftment [9]. On the other hand, it has been shown that MSCs can
survive pro-apoptotic anti-cancer treatments [10]–[11], suggesting that MSCs are
highly resistant to radio-or chemo-induced cell death.

In this study, we have addressed the question of the control of cell death in these
cells. We found that undifferentiated hMSCs were highly resistant to apoptosis while
differentiation, even at the very early stages, was accompanied by an increase in
their sensitivity to apoptosis. This process is differentially controlled by members
of the BCL-2 family of proteins, which are instrumental in the induction of
apoptosis [12] but
have been shown to display distinct roles during hematopoiesis [13].

Materials and Methods

Materials

Unless stated otherwise, all cell culture material was obtained from Gibco
(Invitrogen, Cergy Pontoise, France) and all chemicals were from Sigma-Aldrich
(St. Louis, MO, USA). The 14 bone marrow samples used in this study were
obtained from healthy donors operated at the Dept of Orthopedics at
“Centre Hospitalier Universitaire de Nantes”. The average age of
patients was 41±3 yrs (ranging from 25 to 56 yrs, 6 males and 8 females).
Human fibroblast cultures were obtained from foreskins obtained from the Dept of
Pediatric at “Hôpital Mère et Enfant de Nantes”.

Methods

Ethics statements.

Patient data were obtained and handled according to French laws and
recommendations of the French National Commitee of Ethics (Comité
Consultatif National d'Ethique pour les Sciences de la Vie et de la
Santé).

Cell Culture.

The bone marrow cells were isolated by density gradient centrifugation
(Ficoll). The cells collected at the interface were cultured in alpha-MEM
modified with ribonucleosides and deoxyribonucleosides supplemented with
20% fetal calf serum, with 2 mM L-glutamine, 100 U/mL penicillin, 100
µg/mL streptomycin (complete medium) in an atmosphere of 5%
CO2 and 95% humidity at 37°C. MSC cultures were
used between passages 2 and 10. Cultures were kept at about 75%
confluence and passaged every 5–7 days. Mincing the tissue and trypsin
treatment isolated human foreskin fibroblasts. The fibroblasts recuperated
were cultured in RPMI-1640 supplemented with 10% fetal calf serum, 2
mM L-glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin in an
atmosphere of 5% CO2 and 95% humidity and used
between passages 2 and 35. The K562 cells were cultured in RPMI supplemented
with 10% fetal calf serum, 2 mM L-glutamine, 100 U/mL penicillin, 100
µg/mL streptomycin and Glioma primary cultures (GBM) were cultured in
DMEM supplemented with 10% fetal calf serum, 2 mM L-glutamine, 100
U/mL penicillin, 100 µg/mL streptomycin, both in an atmosphere of
5% CO2 and 95% humidity at 37°C. Cell viability
was determined by Trypan blue exclusion on a minimum of 200 cells using a
Countess automated cell counter (Invitrogen).

Hypoxic treatment.

Cells were cultured in complete medium in an atmosphere of 3%
O2, 5% CO2 and 95% humidity at
37°C in a hypoxia chamber (Invivo 400, 3M, France).

Transfection and viral infection.

hMSCs (106) were nucleofected with 2 µg plasmid: pGFPmax,
pRcCMV (pCMV) empty or containing the Bcl-2 insert (pBcl-2) using the Amaxa
human MSC nucleofector kit (Lonza, Levallois-Perret, France). After 16 h
post-transfection the cells were used in experiments. MSC were cultured with
lentiviral particles (Sigma-Aldrich) at a multiplicity of infection of 15 in
complete medium for 48 h. Lentiviral particles used for the knock-down of
Bcl-Xl were: TRCN0000033499, TRCN0000033500, TRCN0000033501,
TRCN0000033502, TRCN0000033503 and for Nur 77: TRCN0000019425,
TRCN0000019426, TRCN0000019427, TRCN0000019428. The initial experiments were
done with the set of viral particles; however, the repeat experiments were
done with those in bold.

FACScan Analysis.

The phenotype of MSC was monitored by flow cytometry. For phenotypic
analysis, conjugated antibodies were used (cf. Table
S1). Briefly, 2×105 cells were
resuspended in complete medium for 30 min at 4°C. For intracellular
staining, cells were fixed with 4% paraformaldehyde for 10 min and
permeabilized in PBS containing 0,5 % saponin. The cells were
incubated with the primary antibody for 30 min at 4°C in PBS,
0,25% saponin and then, where necessary, the secondary antibody was
added for 30 min at 4°C. Cells were washed twice in PBS before analyzed
on a FACScalibur (BD Biosciences, Le Pont de Claix, France) using Cell Quest
Pro software. The appropriate isotype controls were included and a minimum
of 10 000 events were acquired for each condition. The debris was excluded
from the analysis according to their FSC/SSC proprieties. BD ApoAlert APO
2.7-PE (BD Biosciences) was used to determine the percentage of apoptotic
cells according to the manufacturer's instructions.

Time-lapse analysis.

Time-lapse video-microscopy experiments were performed using a Zeiss Axiovert
200-M inverted microscope (Carl Zeiss, Le Pecq, France) and the AxioVision
4.6 program. Dishes were placed inside an Incubator XL-3, on a heating
insert M06 (37°C) topped with a CO2-cover HM connected to a
CO2 controller that maintained the environmental
CO2 concentration at 5% for the duration of filming.
Digital pictures were acquired and saved every 10 min over 48 h using an
AxioCam MR digital camera. The series of photographs were displayed as
continuous time-lapse movies for analyses. Cell death was quantified at each
acquisition and represented graphically as number of cell death every hour
over the 48 h.

Proximity ligation assay (PLA).

This assay enables the detection and quantification of protein interactions
using secondary antibodies labeled with a pair of oligonucleotides that will
generate a signal when the two PLA probes are in close proximity (Olink
Bioscience, Uppsala, Sweden). The signal is detected as an individual
fluorescent dot. Briefly, the cells were cultured overnight on gelatinized
glass coverslips. Cells were fixed in 4% paraformaldehyde for 15 min,
permeabilized with 0,25% Triton X-100/PBS for 5 min and blocked in
1× blocking solution for 30 min. Cells were incubated with primary
antibodies polyclonal rabbit anti-Nur 77 and monoclonal mouse anti-Bcl-2
diluted in blocking solution overnight at 4°C and then with secondary
antibodies conjugated with oligonucleotides (PLA anti-mouse MINUS and PLA
anti-rabbit PLUS) diluted in blocking solution for 2 h at 37°C. This was
followed by hybridization, ligation, amplification and detection. The
distance between the two primary antibodies must be less than 40 nm to
generate a signal, which results in a highly specific assay to detect for
protein-protein complexes. The slides were mounted using Prolong® Gold
anti-fade reagent with Dapi (Invitrogen) and then analyzed under the
confocal microscope. A 63×1.4 NA oil objective and sequential scanning
with filters 420–480 nm for Dapi and 560–615 nm for fluorophore
Tye-563 were used.

Results

Characterization of hMSCs

MSCs were isolated from human bone marrow samples as described by Pittenger
et al.[14]. After
a few days, the cultures became enriched with fibroblast-like cells that became
predominant after 2 weeks of culture (Figure 1A). The cell population
was characterized by flow cytometric analysis for the expression of CD105, CD90
and CD44 markers (Figure
1B). There was no detectable contamination of
hematopoietic cells since the cells were negative for markers of the
hematopoietic lineage, including the lipo-polysaccharide receptor CD14, CD34,
and the leukocyte common antigen CD45 (Figure 1B). The cells were able
to differentiate into several lineages in vitro, including
osteoblasts and adipocytes (Figures 1C and D). Our findings are in agreement
with previous reports and support the conclusion that our population is enriched
with hMSCs [15]–[16]. Human MSCs cease to
grow after about 20–30 population doublings due to senescence [17]–[18]. The
most common characteristics of a senescent phenotype are a gradual decrease in
the proliferation potential and a decrease in differentiation capacity [19]–[20]. Thus, to avoid the
presence of senescent cells but also that of contaminant or differentiated
cells, we determined that hMSCs should be plated at an initial concentration of
5×104 to 105 and used only between passages 2
and 10.

(A) Cell morphology: Photograph of hMSCs cultured in
complete medium as described in materials
& methods. Note the fibroblast-like morphology of the
cells. (B) Markers (cytometry): Results are represented as
FACScan histograms; the gray line corresponds to the isotype control and
the solid black line to the specific antibody tested. Data shown are
representative of at least three independent experiments. (C,
D) hMSCs were cultured for 3 weeks in either osteogenic
(C) or adipogenic (D) differentiation
media. Matrix mineralization was determined by alkaline phosphatase
staining (C) and lipid vacuoles were stained with Oil Red O
(D) as described in experimental procedures. The
cultures were visualized under an upright Nikon TMS microscope,
magnification ×20.

Undifferentiated hMSCs are resistant to apoptosis

Contrary to the huge body of data available on the control of life and death of
hematopoietic stem cells, very little data are available on the mechanisms of
survival used by hMSCs [21]. It has been shown that MSCs were resistant to
chemotherapy-induced apoptosis [10]–[11], but the actual
mechanism(s) of resistance of these cells to the cell death program were not
determined. Previous data have shown that fetal MSCs are sensitive to both
intrinsic mitochondrial and extrinsic receptor-mediated apoptotic pathways [8]. We thus
examined the response of the isolated hMSCs to various cell death inducers such
as staurosporine (STS), a broad kinase inhibitor, UV-irradiation and etoposide
(Eto), the latter acting mostly through DNA damage-induced apoptosis [22]. As shown
in Figure 2A,
none of these treatments triggered cell death in hMSCs while K562, an
erythroleukemia cell line was sensitive to these cell death inducers. It should
be added that a dose response effect of etoposide and staurosporine was done and
the effect of the optimal dose was used to determine the response to cell death
over time (data not shown). We conclude from these results that
hMSCs are resistant to cell death mediated mainly through the mitochondrial
apoptotic pathway.

(A) Induction of cell death using staurosporine (STS),
UV-irradiation and etoposide (Eto) in K562 cells and hMSCs. The cells
were plated at 5×104 cells/ml in 24-well plates. 24 h
later the different apoptosis inducing agents were added to the culture
medium and the cells were analyzed over 48 h using video-microscopy with
an acquisition every 10 min. The number of dead cells was determined at
each time point and rounded up for every hour. The results are presented
as the percentage of dead cells in treated cultures versus untreated
cultures. The number of cells analyzed was about 100 per condition. The
results are representative of three independent experiments and of 3
different hMSC cultures. (B) Effect of soluble Fas ligand
(sFasL) on hMSCs or GBM primary cultures. Quantification of the
viability of hMSCs cultured in the absence or in the presence of
increasing concentrations (50, 100, 150, 200 and 250 ng/ml) sFasL and
GBM cells cultured in the presence of 50 ng/ml sFasL was determined by
time-lapse microscopy over 24 h using 10 min intervals. Cell viability
was determined at every acquisition as described in (A.
C) Human MSCs or fibroblasts (hfib) were cultured in
the absence or in the presence of etoposide (Eto) at the indicated
concentrations over 24 h. The cells were then lysed and the caspase
activity determined in 20 µg cell extract using Ac-DEVD-AMC as a
substrate. The results are expressed as arbitrary units of caspase
activity per µg protein. (D) Effect of hypoxia on
hMSCs survival. Human MSCs were cultured for 48 h under normoxia
(20% O2) or hypoxia using a hypoxia work-station at
3% O2, 5% CO2 in 95%
humidified air. Note that experiments shown are representative of hMSCs
obtained from at least five different donors.

Next, we investigated the response of these cells to the extrinsic
receptor-mediated apoptotic pathway triggered by soluble Fas ligand (sFasL).
Time-lapse experiments over 24 h showed less than 10% cell death to
concentrations of up to 200 ng/ml sFasL (Figure 2B). Thus, even at very
high concentrations of sFasL, hMSCs did not undergo apoptosis while at a much
lower concentration (i.e. 50 ng/ml) sFasL induced apoptosis in human Glioma
(GBM) primary cultures (Figure 2B), as previously described [23]. Next,
using caspase activity, a landmark of apoptosis, we compare the sensitivity
toward etoposide-induced apoptosis in hMSCs to that of primary cultures of human
foreskin fibroblasts (see experimental procedures). As shown in Figure 2C, hMSCs
were resistant to caspase activation by etoposide while human fibroblasts
exhibited maximum caspase activation at the lowest concentration. As hypoxia and
serum-deprivation induced apoptosis in rat MSCs [24], we examined the effect of
hypoxia on the survival of hMSCs. As shown in Figure 2D, hypoxia did not
induce apoptosis in these cells but on the contrary, it triggered proliferation
as previously described [25].

Lack of cytochrome c release accounts for resistance to apoptosis in
undifferentiated hMSCs

The change in the conformation of Bax, mitochondrial integration and the
subsequent release of cytochrome c (cyt c) are landmarks of apoptosis [12]. As shown in
Figure 3A,
an etoposide-treatment, even at a low concentration (i.e. 50 µg/ml)
triggered an apoptotic change in conformation in Bax in hMSCs as illustrated by
the binding to Bax by the anti-conformational monoclonal antibody 6A7 [12]. The release
of cyt c from mitochondria is a major step in the execution of apoptosis that
occurs downstream of Bax activation and mitochondrial association [12]. Using laser
confocal microscopy as previously described [23], we analyzed the
subcellular localization of cyt c in control and etoposide-treated hMSCs As
illustrated in Figure
3B, no change in the localization of cyt c was observed in
etoposide-treated cells, which remained mitochondrial as observed in control
untreated cells. These results suggest that the blockade of apoptosis in
undifferentiated hMSCs occurred at the mitochondrial level after Bax activation
but before cyt c release. To analyze, the impact of etoposide treatment on hMSCs
growth and differentiation, osteogenic differentiation was induced in control
and etoposide-treated MSC. As shown in Figure 3C, in the absence of
osteogenic differentiation, control and etoposide-treated MSC proliferated at
the same rate for several days (i.e. up to 6 days in vitro).
However, when differentiation was induced after etoposide treatment, hMSCs
stopped proliferating and died after 2 days (Figure 3D). This result suggests
that although undifferentiated hMSCs can sustain heavy DNA damage without the
induction of apoptosis these cells cannot differentiate into mature cells.

Figure 3. Absence of release of cyt c from mitochondria despite the activation
of Bax in hMSCs.

(A, B) Laser confocal analyses of hMSCs after etoposide
treatment. Cells were cultured in the absence (untreated) or in the
presence of 50 µg/ml Etoposide (Eto) for 24 h then labeled with
polyclonal anti-F1-ATPase (mitochondria: green) and either
(A) monoclonal anti-Bax2D2 (2D2: recognizes
all forms of Bax: red) and/or anti-Bax6A7 (6A7: recognizes
only the activated form of Bax: red) or (B) monoclonal
anti-cyt c (red). Laser confocal analyses were done as described in the
material and methods section.
(C, D) Human MSCs were plated into 6-well plates and 24
h later cultured in the absence or presence of 50 µg/ml etoposide
(Eto). 24 h later the cells were either cultured in the presence of
complete media (C) or in osteogenic differentiation media
(D). At the different times indicated the cells were
trypsinized and the number of viable cells determined by trypan blue
exclusion.

The capacity of hMSCs to undergo apoptosis is associated with
differentiation

The latter results suggested that the differentiation process was accompanied by
a change in the sensitivity toward apoptosis. To address this point, hMSCs were
cultured in osteogenic differentiation media for 1 to 3 weeks then treated or
not with etoposide. Cell death was quantified by Trypan blue exclusion 24 h
after the treatment. We found that the initiation of differentiation sensitized
these cells to cell death. Furthermore, as early as one week after the induction
of differentiation, etoposide was able to induce an effective cell death (Figure 4A).
Apoptosis was identified by the measure of caspase activity (Figure 4B) and
analysis with APO 2.7, which both are specific of apoptotic cells [26]
(Figure S1).

Figure 4. Sensitivity to cell death was acquired after induction of
differentiation.

(A) Human MSCs were cultured in complete or osteogenic
differentiation medium for 0 to 3 weeks; in the absence or in the
presence of 50 µg/ml etoposide (Eto). The number of viable cells
was quantified by Trypan blue exclusion counting a minimum of 200 cells
per condition. The data presented represent three independent
experiments. (B) The cells treated as in (A)
were collected and 10 µg whole cell lysates were assayed for
caspase activity using Ac-DEVD-AMC as a substrate. The results are
expressed as arbitrary units of caspase activity per µg protein.
(C) Induction of apoptosis in hMSCs and adipocytes.
Pictomicrographs of adipogenic differentiated hMSCs and hMSCs cultured
in the absence or in the presence of 50 µg/ml etoposide (Eto) for
18 h. Images are representative of 4 independent experiments.
(D) The number of cell death in hMSCs, preadipocyte
(preadipo) or human fibroblast (hfib) cultures treated or not with 50
µg/ml etoposide (Eto) for 24 h was determined by Trypan blue
exclusion (Countess), counting about 200 cells under each condition. The
data are representative of 3 independent experiments. (E)
Human MSCs induced to differentiate along the neuronal pathway (+10
µg/ml BDNF) or not (without BDNF) were cultured in the presence of
50 µg/ml etoposide (Eto) for 24 h before being assayed for caspase
activity using Ac-DEVD-AMC as a substrate. The results are expressed as
arbitrary units of caspase activity per µg protein.

We next investigated the sensitivity of hMSCs toward cell death upon the
induction of differentiation along the adipogenic or neuronal pathways. Cells
were differentiated then subjected to an etoposide treatment. Figure 4C
illustrates cells engaged in adipogenic differentiation treated or not with
etoposide. In agreement with our previous observations, only cells engaged along
the adipogenic differentiation pathway were as sensitive to cell death as human
fibroblasts (Figure
4D). Similarly, hMSCs cultured in the presence of bFGF/EGF and
then BDNF to induce neural differentiation were responsive to etoposide at a
concentration, which did not affect the cells treated only with bFGF/EGF (Figure 4E). Note
that the treatment with etoposide was done in the absence of BDNF since BDNF is
a survival factor and prevents cell death when present in the medium (data
not shown). The decrease in cell viability was due to an increase in
apoptosis as observed by the induction of caspase activity under these
conditions (Figure
4E). Consistent with these results, we found that Bax was
translocated to the mitochondria and cyt c was released from the mitochondria in
etoposide-treated differentiated cells (Figure
S2).

Differential expression of Bcl-2 and Bcl-Xl between undifferentiated and
differentiated hMSCs

The fact that caspase activity could not be detected in undifferentiated hMSCs
upon etoposide, UV or STS treatments whereas Bax was “activated” in
both undifferentiated and differentiated cells suggests that the mitochondrial
apoptotic pathway was impaired in undifferentiated cells at the mitochondrial
level. Thus, we analyzed the expression of key proteins of the apoptotic program
in these cells. As shown in Figure 5A, immunoblots revealed that hMSCs
expressed key components of apoptosis belonging to the BCL-2 family such as the
anti-apoptotic protein Bcl-Xl and the pro-apoptotic protein Bax. Mcl-1 and Bak
were also found in both undifferentiated and differentiated cells (data
not shown). However, undifferentiated hMSCs lack the expression of
Bcl-2. On the other hand, hMSCs also expressed proteins essential for the
execution phase of apoptosis such as caspase 3 (Figure 5A) and caspase 7, 8 and
9 (data not shown). Quite remarkably, Bcl-2 was expressed in
differentiated cells although to a lesser degree in osteoblasts than in
adipocytes (Figure
5A). Indeed, it has been reported that primitive human
hematopoietic precursors (i.e. CD34+/lin−)
express Bcl-Xl but not Bcl-2 [27] and that this differential expression influence their
lineage choice [13].

Figure 5. Expression of key components of the apoptotic machinery in
hMSCs.

(A) Western blot analyses of some of the main components of
the apoptotic machinery. Total protein extracts were performed and 50
µg protein was analyzed on a 12% SDS-PAGE. Immunodetections
were performed with the antibodies cited in the material and methods section. (B) The
sensitivity of the hMSCs-shBcl-Xl-501 and hMSCs-shscr to apoptosis was
determined by culturing the cells in the absence or in the presence of
50 µg/ml etoposide (Eto). The cells were analyzed over 48 h using
video-microscopy with an acquisition every 10 min. The number of dead
cells was determined at each time point and rounded up for every hour.
The results are presented as the percentage of dead cells in treated
cultures versus untreated cultures. The number of cells analyzed was
about 100 per condition. The results are representative of three
independent experiments. (C) Cells were nucleofected with
either pCMV or pBcl-2 and 24 h later cell viability was determined by
Trypan blue exclusion. (D) Twenty-four hours after the
transfection of hMSCs with pCMV (a, b) or pBcl-2 (c, d); these cells
were cultured in the absence (a, c) or presence (b, d) of 50 µg/ml
etoposide (Eto) then analyzed for the expression of active caspase 3
(red) by confocal microscopy. The data presented are representative of 2
independent experiments.

We addressed the question of the involvement of Bcl-Xl and Bcl-2 in the survival
and/or differentiation of hMSCs. First we down-regulated the expression of
Bcl-Xl by shRNA (Figure S3 illustrates the
efficiency of the shRNA Bcl-Xl). The knock-down of Bcl-Xl in hMSCs made these
cells susceptible to low concentrations of etoposide (i.e. 50 µg/ml) that
was not able to trigger apoptosis in hMSCs infected with a scr-shRNA (Figure 5B). This
result suggests that Bcl-Xl plays an essential role in protecting hMSCs against
apoptosis. On the other hand, the over-expression of Bcl-2 (Figure
S4) induced a massive cell death in undifferentiated hMSCs
and this without the addition of apoptotic inducers (Figure 5C). To underline this
observation, hMSCs transfected with Bcl-2, treated or not with etoposide,
exhibited caspase 3 activity while no activity was detected in mock-transfected
cells treated or not with etoposide (Figure 5D).

Bcl-2 induces apoptosis in undifferentiated hMSCs though its interaction with
Nur 77

Recently, Bcl-2 has been shown to be transformed into a pro-apoptotic protein
upon its binding to Nur 77 (also known as TR3 or NGFI-B) through the Bcl-2 loop
region [28]. Nur
77 is present in hMSCs and there is no increase in its expression during
osteogenic differentiation (Figure 6A). The expression of Bcl-2 in
undifferentiated hMSCs but not that of a mutant lacking the binding domain with
Nur 77 (i.e. Bcl-2 loop region) induced apoptosis in these cells (Figure 6B). Data
from the proximity ligation assay (PLA) on hMSCs transfected with either pCMV or
pBcl-2 suggested that Bcl-2 and Nur 77 were in very close proximity to each
other in cells transfected by pBcl-2 as visualized by the number of fluorescent
dots present in these cell (Figure 6C). Nur 77 is present in both the
cytoplasmic and nuclear compartments in undifferentiated hMSCs but is mostly
mitochondrial in hMSCs (Figure S5A). In Bcl-2 transfected
hMSCs, there was a 50% increase in the amount of Nur 77 inserted into the
mitochondria as compared to pCMV-transfected cells (Figure
S5B). A shRNA down-regulation of Nur 77 was performed in
undifferentiated hMSCs (Figure S6). The knock-down of Nur
77 by ShRNA (ShNur 77-25) decreased significantly the Bcl-2-induced cell death
in undifferentiated hMSCs as compared to cells transfected with pCMV (Figure 6D). Note
that a scrambled ShRNA did not affect Bcl-2 induced cell death (Figure 6D).
Similar results were obtained with a ShNur 77 (i.e. ShNur 77-26) that caused no
alteration in Nur 77 expression (data not shown). Taken together,
our results suggest that apoptosis induced by ectopic Bcl-2 in undifferentiated
hMSCs is induced by its interaction with Nur 77.

(A) Western blot analyses of Nur 77 expression in hMSCs,
hMSCs transfected with pBcl-2 and osteoblasts. This is a representative
immunoblot of 2 different experiments. (B) Cells were
transfected with plasmids: pCMV, pCMV containing Bcl-2 (pBcl-2) or Bcl-2
deleted in the amino acids 30–85 (pBcl-2Δloop). 16 h
post-transfection the cells were plated in 6-well plated and
video-filmed over 48 h with acquisition every 10 min. The number of dead
cells was determined at each time point and rounded up for every hour.
The results are presented as the percentage of dead cells in treated
cultures versus untreated cultures. The number of cells analyzed was
minimum 100 per condition. The results are representative of 3
independent experiments. (C) A PLA between Bcl-2 and Nur 77
was performed on hMSCs transfected with pCMV or pBcl-2 as described in
the materials and methods section.
Fluorescence was detected using the fluorophore Tye-563 that excites at
557 nm and emits at 563 nm. The nuclei were stained with Dapi present in
the mounting solution (Prolong® Gold anti-fade). Spots indicated
interaction between Nur77 and Bcl-2. These experiments were repeated 3
times, each performed in duplicate. (D) Human MSCs infected
with either shNur77 or shscr were then transfected with pCMV or pBcl-2
and then treated with 50 µg/ml etoposide. Note that for all
experiments only Nur77-25 was used. Cell viability was determined by
Trypan blue exclusion, 24 h after treatments. The results are
representative of 3 different experiments done in triplicate.

Discussion

In this study, we have analyzed the relationship between sensitivity to cell death
and differentiation of hMSCs. We have found that undifferentiated hMSC were highly
resistant to apoptosis until they engaged along a differentiation pathway. The hMSCs
used in this study were isolated from middle-aged donors; were short-term cultures
and used for a limited number of passages to avoid the interference with senescence
(Figure 1). In
this study, we show that hMSCs are resistant to potent apoptosis inducers at
concentrations that otherwise kill cancer cells such as the K562 cells or human GBM
cells as well as primary cultures of human fibroblasts (Figure 2). The nature of this
intrinsic resistance to apoptosis implies the inhibition of the mitochondrial
intrinsic pathway as demonstrated by the lack of cyt c release upon the induction of
apoptosis in hMSCs (Figure
3). Human MSCs that have undergone DNA damage during etoposide
treatment, survive and proliferate but die upon differentiation (Figure 3). However,
it should be noted that this treatment might not reflect the type of DNA damage that
could occur under normal physiological conditions. However, one could postulate that
these features are involved in the elimination of damaged hMSCs. Of note, upon
addition of differentiating factors, hMSCs shift their sensitivity toward apoptosis
inducers (Figure
4). This feature is not restricted to osteogenic lineage as it can be
also observed during neural and adipocytic differentiation as well (Figure 4).

One important finding in this work is the differential expression of Bcl-2 between
differentiated and undifferentiated hMSCs (Figure 5). The control of Bcl-2
versus cell differentiation has been shown in several cell types, including
hematopoietic, neural and epithelial cells [13]. Low levels of Bcl-2 are found
in immature cells and high levels in mature cells in the lymphoid compartment [29]. Similarly,
Bcl-2 expression is up-regulated during the differentiation of hematopoietic
progenitors. Our results strengthen the hypothesis of a link between low Bcl-2
levels and “stemness” outside the hematopoietic compartment. In hMSCs
this effect appears to be different from its anti-apoptotic role, which is
apparently carried out by Bcl-Xl (Figure 5). The latter feature is consistent with
previous studies that show that Bcl-Xl functions as a prime regulator of the
viability of immature cells during the development of the nervous and hematopoietic
system [30].
However, in this study we have analyzed the behavior of isolated hMSCs, a condition
that does not necessarily mirror a physiological situation.

On the other hand, Bcl-2 induces cell death when expressed in undifferentiated hMSCs
though its interaction with Nur 77 (Figure 6). Nur 77 is an orphan member of the nuclear
receptor subfamily 4 that is expressed in many cell types where it plays several key
roles including induction of apoptosis and control of differentiation. Inhibition of
differentiation by Nur 77 through its transcriptional targets has been documented in
adipocytes [31]. On
the other hand, it has been shown to induce cell cycle arrest and differentiation in
dopaminergic cells [32]. During TCR-mediated clonal deletion, Nur 77 appears to
be instrumental through its capacity to induce apoptosis. It is thought that the
translocation of Nur 77 to the cytoplasm promotes cell death, while its retention in
the nucleus promotes survival and proliferation through transcriptional activity but
also via protein-protein interactions. Two of Nur 77 primary interactive targets are
Bcl-2 and Bcl-B, the binding to which induces a drastic change, switching their
function from anti-apoptotic to pro-apoptotic [28]. However, Nur 77 is not capable of
interacting with either Bcl-Xl or Mcl-1 [33]. The interaction between Nur
77 and Bcl-2 is instrumental in the induction of apoptosis during negative selection
of autoreactive thymocytes [34]. Here, we show a parallel mechanism in hMSCs,
demonstrating that Bcl-2 is down-regulated in undifferentiated hMSCs and this
down-regulation preserves these cells from cell death via apoptosis. These results
suggest that a complex cross-regulation between Bcl-2 and Nur 77 controls the
survival and the differentiation of hMSCs through a mechanism similar to that
observed during the negative selection in the immune system [35].

A better understanding of the different mechanisms that regulate apoptosis in hMSCs
could provide important clues on the relationship between the cell death program and
the differentiation programs in adult stem cells. This could have important
consequences on both cancer and regenerative medicine.

Supporting Information

Human MSCs were cultured in complete or osteogenic differentiation
medium for 0, 1 or 3 weeks and in the absence or presence of 50
µg/ml etoposide (Eto). The cells were trypsinized and the
number of apoptotic cells was labelled with APO 2.7-PE and then quantified
by cytometry. The results are representative of three independent
experiments.

(A) The localization of Nur 77 in hMSCs nucleofected with pBcl-2
or pCMV was determined by confocal microscopy. These are representative
pictures of 3 different experiments. (B) Calculation of the
percentage of co-localization of Nur 77 (green) with F1-ATPase
(mitochondria: red) in hMSCs transfected with either pCMV or pBcl-2 using
Metamorph 7.5.6. The algorithm «XOR» was applied to Nur 77 and
the «AND» algorithm was used to threshold the non-colocalized
Nur 77. The results are the mean of 10 views from 3 different
experiments.

Western blot analyses of hMSCs infected with sh-scr or shNur 77 showing
the different levels of knock-down of Nur 77 using the different shNur
77 viral particles. Note for the experiments Nur 77-25 was
used.